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Protein identification

Confirmation of protein primary sequences and intact protein mass

We use multi-enzyme digestion strategies combined with high resolution MS to confirm the primary sequence of a protein, and we determine its molecular weight using intact protein MS.

Identification of the proteins and estimation of their relative amounts in a sample

We use nano-LC-MS/MS to identify the proteins present in a given sample (e.g. purified protein/protein complex, gel band, IP, BioID, organelle, cell, tissue, biofluid). We estimate the % contribution of each protein to the total protein content detected in the sample.

Identification of post-translational modifications (PTMs)

We use nano-LC-MS/MS with and without specific enrichment of modified peptides to identify PTMs and modified amino acids within a protein sequence (e.g. phosphorylation, Lys-acetylation, ubiquitination, proteolytic cleavage by endogenous proteases).

Services We Provide

Quantitative proteomics

Relative and unbiased quantitation of 1000s of proteins

We use label free quantitation (LFQ), isobaric labeling techniques (iTRAQ, TMT), as well as SILAC to relatively quantify 1000s of proteins across multiple samples.

Relative and unbiased quantitation of PTMs (e.g. phosphorylation, ubiquitination, Lys-acetylation, proteolytic cleavage)

We use label free quantitation (LFQ), isobaric labeling techniques (iTRAQ, TMT), as well as SILAC in combination with methods for the enrichment of modified peptides (e.g. IMAC, antibodies against Lys-acetyation) to relatively quantify 1000s of PTMs across multiple samples.

Highly multiplexed ‘absolute’ quantitation of selected protein targets and PTMs

We use targeted mass spectrometry (multiple reaction monitoring, MRM, and parallel reaction monitoring, PRM) for the highly sensitive, highly specific, and highly precise (CVs <20%) ‘absolute’ quantitation (i.e. determining the concentration) of proteins and PTMs. We have developed MRM assays for thousands of proteins and can run multiplexed assays to quantify >200 targets within a single analysis. These assays are ideal for validation purposes and for screening hundreds of samples.

Metabolomics

Development of custom metabolite assays

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We develop and perform LC-MS analyses for wide range of the metabolites, exogenous substances, and pharmaceuticals.

Quantitative targeted and untargeted metabolite profiling

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We perform targeted (MRM-based LC-MS analysis of the pathway- and class-specific metabolite panels) and untargeted (LC-MS analysis) metabolite profiling in biological samples.

Specialized metabolomics assays (13C-fluxome analysis, MetID)

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We develop and perform custom specialized metabolic assays using isotopically-labeled metabolite analogs (13C-labeled metabolome tracing fluxome analysis, comprehensive identification of the drug metabolites in primary hepatocyte cultures and animal models).

Clinical MS

Development of custom disease-specific targeted proteomics and metabolomics assays

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We develop, validate and implement into clinical practice disease-specific proteomics and metabolomics assay panels.

Development of custom clinical chemistry and TDM LC-MS

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We develop, validate and implement into clinical practice custom LC-MS assays for the determination of the analytes of clinical chemistry interest and the therapeutic drug monitoring assays.

Development of immuno-MS assays for quantitation of selected protein target concentrations with ultra-high sensitivity

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We develop highly sensitive and precise assays for determining the concentration of selected protein targets in cells, tissues (fresh-frozen and FFPE), as well as biofluids. For this purpose we combine immuno-enrichment using custom-made anti-peptide antibodies with MS. The antibodies are directed against a ‘proteotypic’ surrogate peptide of the target protein that is released upon proteolytic digestion of a given sample. Spiking-in a known amount of a stable isotope labeled standard (SIS) peptide that has the exact same amino acid sequence as the target peptide, allows the ‘absolute’ quantitation of the target protein using either multiple reaction monitoring (iMRM) or MALDI (iMALDI) mass spectrometry. This allows quantifying as little as 100 amol of protein from 10 µg of protein extract with high precision and reproducibility with robust mass spectrometry methods (i.e. microflow LC-MRM and MALDI).

Structural proteomics

Structural characterization of protein therapeutics and biosimilars (intact mass, correct folding, aggregation state, PTMs)

  • Mapping protein-protein interaction interfaces and antibody epitope determination

We use exact intact protein mass determination and intact mass, bottom-up and top-down hydrogen-deuterium exchange for the structural characterization of the protein therapeutics and the structural comparison of the protein biosimilars.

Identification of protein drug targets and determination of drug-binding sites

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We use a combination of the affinity enrichment techniques, photo-affinity labeling and hydrogen-deuterium exchange for the determination and characterization of the small molecule drug target proteins and drug binding sites.

Mapping protein-protein interaction interfaces and antibody epitope determination

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We use a combination of the crosslinking, covalent surface modification, hydrogen-deuterium exchange and limited proteolysis for the comprehensive identification and characterization of the protein interaction interfaces.

de novo solving protein structures

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We use a combination of the structural proteomics techniques (short-distance crosslinking, photo-reactive surface modification, hydrogen-deuterium exchange, limited proteolysis) and molecular dynamics simulations for the solving unknown protein structures.

Determining topology of the protein complexes

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We use long-distance crosslinking for the determination of the topology of the multi-subunit protein assemblies.

Identification of protein-protein interaction on proteome-wide level  

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We use crosslinking for the determination of the protein interaction networks in the native cell and tissue environments.

Protein identification

Identification of the proteins and estimation of their relative amounts in a sample

  1. Histone demethylation by a family of JmjC domain-containing proteins. Tsukada Y, Fang J, Erdjument-Bromage H, Warren ME, , Tempst P, Zhang Y. . 2006 Feb 16;439(7078):811-6.

  2. Purification and functional characterization of a histone H3-lysine 4-specific methyltransferase. Wang H, Cao R, Xia L, Erdjument-Bromage H, , Tempst P, Zhang Y. . 2001 Dec;8(6):1207-17.

Identification of post-translational modifications (PTMs)

  1. JNK phosphorylates paxillin and regulates cell migration. Huang C, Rajfur Z, Borchers C, Schaller MD, Jacobson K. Nature. 2003 Jul 10;424(6945):219-23.

  2. Global analysis of the mitochondrial N-proteome identifies a processing peptidase critical for protein stability. Vögtle FN, Wortelkamp S, Zahedi RP, Becker D, Leidhold C, Gevaert K, Kellermann J, Voos W, Sickmann A, Pfanner N, Meisinger C. Cell. 2009 Oct 16;139(2):428-39.

  3. Inhibition of osimertinib-resistant epidermal growth factor receptor EGFR-T790M/C797S. Lategahn J, Keul M, Klövekorn P, Tumbrink HL, Niggenaber J, Müller MP, Hodson L, Flaßhoff M, Hardick J, Grabe T, Engel J, Schultz-Fademrecht C, Baumann M, Ketzer J, Mühlenberg T, Hiller W, Günther G, Unger A, Müller H, Heimsoeth A, Golz C, Blank-Landeshammer B, Kollipara L, Zahedi RP, Strohmann C, Hengstler JG, van Otterlo WAL, Bauer S, Rauh D. Chem Sci. 2019 Oct 4;10(46):10789-10801.

  4. Effective Assignment of α2,3/α2,6-Sialic Acid Isomers by LC-MS/MS-Based Glycoproteomics. Pett C, Nasir W, Sihlbom C, Olsson BM, Caixeta V, Schorlemer M, Zahedi RP, Larson G, Nilsson J, Westerlind U. Angew Chem Int Ed Engl. 2018 Jul 20;57(30):9320-9324.

Quantitative proteomics

Relative and unbiased quantitation of 1000s of proteins

  1. Landscape of submitochondrial protein distribution. Vögtle FN, Burkhart JM, Gonczarowska-Jorge H, Kücükköse C, Taskin AA, Kopczynski D, Ahrends R, Mossmann D, Sickmann A, Zahedi RP, Meisinger C. Nat Commun. 2017 Aug 18;8(1):290.

The first comprehensive and quantitative analysis of human platelet protein composition allows the comparative analysis of structural and functional pathways. Burkhart JM, Vaudel M, Gambaryan S, Radau S, Walter U, Martens L, Geiger J, Sickmann A, . . 2012 Oct 11;120(15):e73-82.

Relative and unbiased quantitation of PTMs (e.g. phosphorylation, ubiquitination, Lys-acetylation, proteolytic cleavage)

  1. Proteome-wide detection of S-nitrosylation targets and motifs using bioorthogonal cleavable-linker-based enrichment and switch technique. Mnatsakanyan R, Markoutsa S, Walbrunn K, Roos A, Verhelst SHL, Zahedi RP. Nat Commun. 2019 May 16;10(1):2195. doi: 10.1038/s41467-019-10182-4. PMID: 31097712

  2. PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis. Saita S, Nolte H, Fiedler KU, Kashkar H, Venne AS, Zahedi RP, Krüger M, Langer T. Nat Cell Biol. 2017 Apr;19(4):318-328. doi: 10.1038/ncb3488. Epub 2017 Mar 13. PMID: 28288130

  3. Temporal quantitative phosphoproteomics of ADP stimulation reveals novel central nodes in platelet activation and inhibition. Beck F, Geiger J, Gambaryan S, Solari FA, Dell'Aica M, Loroch S, Mattheij NJ, Mindukshev I, Pötz O, Jurk K, Burkhart JM, Fufezan C, Heemskerk JW, Walter U, Zahedi RP, Sickmann A. Blood. 2017 Jan 12;129(2):e1-e12. doi: 10.1182/blood-2016-05-714048. Epub 2016 Nov 9. PMID: 28060719

  4. Amyloid-β peptide induces mitochondrial dysfunction by inhibition of preprotein maturation. Mossmann D, Vögtle FN, Taskin AA, Teixeira PF, Ring J, Burkhart JM, Burger N, Pinho CM, Tadic J, Loreth D, Graff C, Metzger F, Sickmann A, Kretz O, Wiedemann N, Zahedi RP, Madeo F, Glaser E, Meisinger C. Cell Metab. 2014 Oct 7;20(4):662-9. doi: 10.1016/j.cmet.2014.07.024. Epub 2014 Aug 28. PMID: 25176146

Highly multiplexed ‘absolute’ quantitation of selected protein targets and PTMs

  1. Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma. Addona TA, Abbatiello SE, Schilling B, Skates SJ, Mani DR, Bunk DM, Spiegelman CH, Zimmerman LJ, Ham AJ, Keshishian H, Hall SC, Allen S, Blackman RK, , Buck C, Cardasis HL, Cusack MP, Dodder NG, Gibson BW, Held JM, Hiltke T, Jackson A, Johansen EB, Kinsinger CR, Li J, Mesri M, Neubert TA, Niles RK, Pulsipher TC, Ransohoff D, Rodriguez H, Rudnick PA, Smith D, Tabb DL, Tegeler TJ, Variyath AM, Vega-Montoto LJ, Wahlander A, Waldemarson S, Wang M, Whiteaker JR, Zhao L, Anderson NL, Fisher SJ, Liebler DC, Paulovich AG, Regnier FE, Tempst P, Carr SA. . 2009 Jul;27(7):633-41.

  2. Multiplexed targeted proteomic assay to assess coagulation factor concentrations and thrombosis-associated cancer.

  3. Mohammed Y, van Vlijmen BJ, Yang J, Percy AJ, Palmblad M, , Rosendaal FR. . 2017 Jun 20;1(15):1080-1087.

  4. Multiplex quantitation of 270 plasma protein markers to identify a signature for early detection of colorectal cancer.

  5. Bhardwaj M, Weigl K, Tikk K, Holland-Letz T, Schrotz-King P, , Brenner H. . 2020 Mar;127:30-40.

  6. Mouse Quantitative Proteomics Knowledgebase: reference protein concentration ranges in 20 mouse tissues using 5000 quantitative proteomics assays. Mohammed Y, Bhowmick P, Michaud SA, Sickmann A, . . 2021 Jan 23:btab018.

Clinical MS

Development of custom disease-specific targeted proteomics and metabolomics assays

  1. Development and evaluation of an immuno-MALDI (iMALDI) assay for angiotensin I and the diagnosis of secondary hypertension. Camenzind AG, van der Gugten JG, Popp R, Holmes DT, Borchers CH. Clin Proteomics. 2013 Dec 20;10(1):20.

  2. An LC-MRM assay for the quantification of metanephrines from dried blood spots for the diagnosis of pheochromocytomas and paragangliomas. Richard VR, Zahedi RP, Eintracht S, . Anal Chim Acta. 2020 Sep 1;1128:140-148.

  3. Metabolic profiling of bile acids in human and mouse blood by LC-MS/MS in combination with phospholipid-depletion solid-phase extraction. Han J, Liu Y, Wang R, Yang J, Ling V, . Anal Chem. 2015 Jan 20;87(2):1127-36.

  4. MRM-based multiplexed quantitation of 67 putative cardiovascular disease biomarkers in human plasma. Domanski D, Percy AJ, Yang J, Chambers AG, Hill JS, Freue GV, . Proteomics. 2012 Apr;12(8):1222-43.

  5. Short-Term Stabilities of 21 Amino Acids in Dried Blood Spots. Han J, Higgins R, Lim MD, Lin K, Yang J, . Clin Chem. 2018 Feb;64(2):400-402.

  6. Concentration Determination of >200 Proteins in Dried Blood Spots for Biomarker Discovery and Validation. Eshghi A, Pistawka AJ, Liu J, Chen M, Sinclair NJT, Hardie DB, Elliott M, Chen L, Newman R, Mohammed Y, . Mol Cell Proteomics. 2020 Mar;19(3):540-553.

Development of custom clinical chemistry and TDM LC-MS assays

  1. Direct and Precise Measurement of Bevacizumab Levels in Human Plasma Based on Controlled Methionine Oxidation and Multiple Reaction Monitoring. Spatz A, ACS Pharmacol Transl Sci. 2020 Nov 13;3(6):1304-1309.

Development of immuno-MS assays for quantitation of selected protein target concentrations with ultra-high sensitivity

  1. Duplexed iMALDI for the detection of angiotensin I and angiotensin II. Mason DR, Reid JD, Camenzind AG, Holmes DT, Borchers CH. Methods. 2012 Feb;56(2):213-22.

  2. Immuno-Matrix-Assisted Laser Desorption/Ionization Assays for Quantifying AKT1 and AKT2 in Breast and Colorectal Cancer Cell Lines and Tumors. Popp R, Li H, LeBlanc A, Mohammed Y, Aguilar-Mahecha A, Chambers AG, Lan C, Poetz O, Basik M, Batist G, Borchers CH. Anal Chem. 2017 Oct 3;89(19):10592-10600.

  3. Immuno-MALDI (iMALDI) mass spectrometry for the analysis of proteins in signaling pathways. Popp R, Li H, Borchers CH. Expert Rev Proteomics. 2018 Sep;15(9):701-708.

  4. How iMALDI can improve clinical diagnostics. Popp R , Basik M , Spatz A , Batist G , Zahedi RP , Borchers CH. Analyst. 2018 May 15;143(10):2197-2203.

Structural proteomics

  1. Architecture of the RNA polymerase II-Mediator core initiation complex. Plaschka C, Larivière L, Wenzeck L, Seizl M, Hemann M, Tegunov D, Petrotchenko EV, Borchers CH, Baumeister W, Herzog F, Villa E, Cramer P. Nature. 2015 Feb 19;518(7539):376-80.

  2. Recommendations for performing, interpreting and reporting hydrogen deuterium exchange mass spectrometry (HDX-MS) experiments. Masson GR, Burke JE, Ahn NG, Anand GS, Borchers C, Brier S, Bou-Assaf GM, Engen JR, Englander SW, Faber J, Garlish R, Griffin PR, Gross ML, Guttman M, Hamuro Y, Heck AJR, Houde D, Iacob RE, Jørgensen TJD, Kaltashov IA, Klinman JP, Konermann L, Man P, Mayne L, Pascal BD, Reichmann D, Skehel M, Snijder J, Strutzenberg TS, Underbakke ES, Wagner C, Wales TE, Walters BT, Weis DD, Wilson DJ, Wintrode PL, Zhang Z, Zheng J, Schriemer DC, Rand KD. Nat Methods. 2019 Jul;16(7):595-602.

  3. Discovery of a small-molecule HIV-1 integrase inhibitor-binding site. Al-Mawsawi LQ, Fikkert V, Dayam R, Witvrouw M, Burke TR Jr, Borchers CH, Neamati N. Proc Natl Acad Sci U S A. 2006 Jun 27;103(26):10080-5.

  4. Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem-loop-binding proteins that contributes to high-affinity RNA binding. Borchers CH, Thapar R, Petrotchenko EV, Torres MP, Speir JP, Easterling M, Dominski Z, Marzluff WF. Proc Natl Acad Sci U S A. 2006 Feb 28;103(9):3094-9.

  5. Solving protein structures using short-distance cross-linking constraints as a guide for discrete molecular dynamics simulations. Brodie NI, Popov KI, Petrotchenko EV, Dokholyan NV, Borchers CH. Sci Adv. 2017 Jul 7;3(7):e1700479.

  6. Protein unfolding as a switch from self-recognition to high-affinity client binding. Groitl B, Horowitz S, Makepeace KAT, Petrotchenko EV, Borchers CH, Reichmann D, Bardwell JCA, Jakob U. Nat Commun. 2016 Jan 20;7:10357.

CONTACT

Segal Cancer Proteomics Centre
E615
3755 Chemin de la Cote-Sainte-Catherine
Montreal, QC, H3T 1E2 Canada

Tel: +1 (514) 340-8222 ext: 27885
 

Segal Cancer Proteomics Centre

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